The evolution of enzymes was a critical element of the emergence of life on Earth and its subsequent divergence to occupy a vast range of ecological niches. Most enzymes have impressive catalytic capabilities that have been honed over 3.8 billion years of evolution. We are interested in the early stages of evolution of new enzymes. This application addresses enzymes that appear to have been recently recruited to serve new functions in the pathway for degradation of pentachlorophenol (PCP) in Sphingobium chlorophenolicum, a bacterium isolated from soil heavily contaminated with PCP. S. chlorophenolicum appears to have patched together a poorly functioning pathway for degradation of PCP using enzymes from at least two previously existing pathways. This experimental system provides us with a window on the evolution of a new metabolic pathway at a very early stage in the process. This application focuses on the initial three enzymes in the pathway that limit the flux through the PCP degradation pathway. PCP hydroxylase catalyzes the hydroxylation of PCP to form the toxic intermediate, tetrachlorobenzoquinone (TCBQ). TCBQ reductase catalyzes the reduction of TCBQ to tetrachlorohydroquinone (TCHQ). TCHQ dehalogenase catalyzes two successive reductive dehalogenation reactions that remove chlorines, allowing the ring to be cleaved. Our aims are 1) to obtain structures of PCP hydroxylase and TCHQ dehalogenase;2) to explore the evolutionary origin and a possible additional function of TCBQ reductase;3) to evolve improved versions of all three enzymes;and 4) to determine how the improved enzymes affect the ability of the bacterium to degrade PCP. The outcome of this work will be a better understanding of the sources from which new enzymes can arise, and the quality of catalysis achieved during the initial stages of evolution of a new enzyme. PUBLIC HEALTH RELEVANCE: We will better understand the prospects for evolution of pathways to degrade toxic pollutants and the problems associated with development of antibiotic resistance by recruitment of enzymes that detoxify antibiotics. In addition, this work will inform efforts to engineer enzymes and bacteria to produce chemicals and pharmaceuticals or to degrade pollutants under environmentally benign conditions.